Figure 3. The MYC–host cell factor (HCF)–1 interaction influences the expression of genes involved in ribosome biogenesis and mitochondrial pathways.

Switchable Ramos cells were treated with 20 nM 4-OHT for 24 hr, RNA isolated, and RNA-Seq performed. (A) Scatterplot showing the distribution of log₂FC of significant (false discovery rate [FDR] < 0.05) RNA-Seq changes with the 4A and VP16 HCF-1-binding motif (HBM) MYC mutants, compared to the wild-type (WT) switch. Solid lines represent the median log₂FC for decreased (4A: −0.2858; VP: −0.2747) and increased (4A: 0.281; VP: 0.2558) transcripts compared to WT. For clarity, some data points were excluded; these data points are highlighted in Figure 3—source data 1 and 2. (B and C) Categories from the top eight families in gene ontology (GO) enrichment analysis of significant (FDR < 0.05) gene expression changes under each condition (B: 4A; C: VP16 HBM). (D) Heatmap showing the log₂FC of significantly (FDR < 0.05) changed transcripts that are altered in expression in both the 4A and VP16 HBM mutants. Transcripts are clustered according to the relationship in expression changes between the 4A and VP16 HBM mutants, and ranked by the log₂FC for the 4A mutant. Scale of heatmap is limited to [−3,3]. (E and F) Gene clusters in (D) were subject to GO enrichment analysis, and the top eight categories are shown for the correlative (E) or anti-correlative (F) clusters. The Q-value of categories is represented by bubble color, and the number of genes present in a category is represented by bubble size. The genes in these categories are identified in Figure 3—figure supplement 2A and B.

Figure 3—figure supplement 1. Gene expression changes induced by the 4A and VP16 host cell factor (HCF)–1-binding motif (HBM) mutants.

(A) Volcano plots showing the significant (false discovery rate [FDR] < 0.05) gene expression changes for the 4A (left) and VP16 HBM (right) mutants. For clarity, some data points were excluded; these data points are highlighted in Figure 3—source data 1 and 2. (B) Heatmap visualizing consistency among replicates of RNA-Seq for switchable MYC allele cell lines at 24 hr, by z-transformation and ranking by fold-change (FC). Transcripts that were significantly (FDR < 0.05) impacted compared to wild type (WT) are shown. Three biological replicates for each WT, 4A, and VP16 HBM were used to calculate FDR and FCs. (C) Validation of a set of gene expression changes detected in RNA-Seq. Cells were grown with or without 4-OHT for 24 hr, RNA isolated, reverse transcribed, and the indicated transcripts quantified by real-time PCR. Cycle threshold (Ct) values were normalized to those for GAPDH and FCs calculated over −4-OHT samples. Shown are the mean and standard error for three biological replicates. Student’s t-test between WT and mutant cells was used to calculate p-values; *p<0.05, **p<0.01, ***p<0.001. (D) Significant (FDR < 0.05) gene expression changes that are anti-correlative (top) and correlative (bottom) in direction between the 4A and VP16 HBM mutants.

Figure 3—figure supplement 2. Gene ontology (GO) enrichment analysis of correlative and anti-correlative effects on gene expression.

(A and B) Categories from the top eight families in GO enrichment analysis of the anti-correlated (A) and correlated (B) gene clusters shown in Figure 3D, for transcripts that were decreased (top) or increased (bottom) with the 4A mutant. The Q-value of categories is represented by the ribbon color, which is scaled across these figures. Categories are ranked by the number of matched genes, and genes are ranked by the number of categories into which they fall.

Figure 3—figure supplement 3. Amino acids and their cognate tRNA-ligases are influenced by the MYC–host cell factor (HCF)–1 interaction.

Impact of the 4A (A) and VP16 HCF-1-binding motif (B) mutants on amino acid levels (hydrophilic interaction liquid chromatography) and on the expression of aminoacyl tRNA transferases (RNA-Seq).